PROJECT SUMMARY/ABSTRACT Mechanical forces within the material microenvironment are increasingly recognized as important regulators of stem cell self-renewal and differentiation. Over the past decade we have been exploring these concepts in the context of adult hippocampal neural stem cells (NSCs), which generate neurons throughout adult life and play key roles in learning, memory, and disease processes. In our first period of R01 support, we have shown that extracellular matrix (ECM) stiffness cues can act through Rho GTPase- and myosin-dependent contractility to influence lineage commitment within a defined temporal window. Moreover, manipulation of these stiffness- sensing pathways in vivo can control hippocampal neurogenesis in a manner that is predictable from culture studies. More recently, we have created reversibly-stiffening oligonucleotide-crosslinked materials and applied this technology to narrow this window to 12-36 h and to begin elucidating key signals that are activated during this period to induce lineage commitment. In this renewal application, we now propose to build upon these advances by tackling two key questions of high general interest within the stem cell field: First, do the mechanoregulatory signaling relationships observed in simplified 2D systems hold in more complex 3D microenvironments, particularly ones with dynamic mechanical properties analogous to those encountered in vivo? Second, precisely how do the signals triggered by mechanical inputs (e.g. Rho GTPase-dependent myosin contraction) interface with the signals canonically understood to regulate NSC neurogenesis? In Aim 1, we will investigate mechanosensitive lineage commitment in 3D by applying new click-crosslinked hyaluronic acid hydrogels with tunable stiffness. We will also innovate upon these materials by incorporating reversible oligonucleotide-based crosslinks that allow variable degrees of stress relaxation, and then use these materials to ask if we can shift the time window of mechanosensitive lineage commitment. In Aim 2, we will investigate integration of mechanotransductive signaling and canonical pro-neurogenic signaling in the control of NSC neurogenesis. Specifically, we will test the hypothesis that mechanosensitive lineage commitment is controlled by a master signaling circuit involving YAP, angiomotin, and b-catenin. We will also apply genome-wide CRISPR gain/loss-of-function screens to identify additional candidates, which we will then characterize and incorporate into this regulatory framework. Successful completion of these studies will not only dramatically improve the field?s understanding of how mechanical signals influence NSC lineage commitment but offer a new intellectual roadmap and set of tools that will be broadly applicable to all stem cell types.